Herpes simplex virus (HSV) encodes 60-70 functions, most of which regulate productive replication, but some of which probably control the establishment and maintenance of latency and transformation. Due to the structural complexity of HSV DNA, it is likely that the genome encodes many functions required for viral DNA replication. Temperature-sensitive (ts) mutants would provide a means of identifying these functions. However, to date the DNA- mutant, ts D9, is the only ts mutant found to be defective in a protein directly involved in HSV DNA synthesis (i.e., DNA polymerase). The other characterized DNA-mutants are defective in regulatory functions which prevent DNA synthesis. The DNA plus or minus ts mutants also are defective in DNA synthesis although these have not been studied in detail. In an effort to obtain a means by which DNA synthesis can be studied effectively, we will attempt to identify ts mutants with defects in structural genes involved in DNA synthesis. Ts mutants will be obtained by mutagenesis of specific DNA sequences and transferred to intact genomes by recombination. The DNA produced at the nonpermissive temperature by DNA plus or minus ts mutants will be analyzed for sequences present, size, degree of nicking and infectivity to determine whether it represents mature, intact, fully processed DNA. RNA transcribed from DNA regions containing the ts lesion will be translated in vitro. Translation products as well as extracts from infected cells will be assayed for possible enzymatic activities associated with DNA synthesis.